RNA interference (RNAi) is a recently discovered gene-silencing phenomenon mediated by double stranded short interfering RNA (siRNA) that guide mRNA degradation in a sequence specific fashion. We have shown that siRNAs targeting cellular and viral genes involved in HIV life cycle can dramatically suppress virus replication in human cell lines and primary cells. Since RNAi is critically dependent on nucleotide sequence match, the propensity of HIV for sequence mutations is an impediment for using the technology as therapy against the virus. To overcome this limitation, we have identified optimal siRNA targets in highly conserved regions of the HIV genome and showed that these shRNAs can protect primary CD4 T cells from HIV-1 primary isolates within clade B or across multiple clades. In vivo delivery of siRNA/shRNA is the other major hurdle for translation of this promising new technology into the clinic. To use siRNA as a drug it will be critical to develop a method to introduce siRNA into primary T cells and macrophages. This is a major challenge because T cells take up siRNA very poorly even by in vitro transfection. We have recently shown that targeted delivery of siRNA to this difficult cell type can be accomplished by using a single chain antibody fused to protamine, which is a highly basic polypeptide that can bind siRNA. Further, we have also used a novel immunoliposome conjugated to 2 different antibodies that recognize LFA-1 either in the closed or open activated conformation to differentially deliver siRNA to all T cells irrespective of the activation status or specifically to activated T cells only. One other problem in a chronic infection like HIV is the need for long-term therapy. To this end, we have developed lentiviral vectors to endogenously express shRNAs and showed its potential to derive HIV-resistant progeny by transduction of CD34+ hematopoietic stem cells (HSCs). We now propose to optimize delivery methods for potential RNAi-based interventions in HIV infection in a humanized mouse model, using the novel SCID/NODIL2r?c-/- mouse strain developed by our collaborator Dr. Leonard Shultz at the Jackson Laboratory. The common gamma chain null SCID/Hu mice support complete development of human immune system after engraftment with purified CD34+ hematopoietic stem cells and reconstituted animals infected with HIV-1 show sustained and high levels of viral replication. Thus, the model provides us with a unique experimental system in which to optimize our delivery strategies. Our specific objectives are first to further refine the antibody/protamine fusion protein- and immunoliposome- mediated naked siRNA delivery to resting and/or activated T cells using antibodies to CD7 (a pan T cell molecule) or LFA-1 in open or closed conformation. Our next goal will be to test the in vivo efficacy of both delivery strategies in NOD/Lt-scid IL2r?null mice humanized either by PBL transplantation (SCID/hu-PBL) or by engrafting with cord blood CD34+ hematopoietic stem cells. Finally, we will reconstitute NOD/Lt-scid IL2r?null mice with lentivirus-transduced CD34+ human HSCs and test establishment of long-term HIV resistance.